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Prof. Neil Jones rnj@aber.ac.uk

Barbara McClintock’s controlling elements: the full story. Prof. Neil Jones rnj@aber.ac.uk. IBERS - Institute of Biological, Environmental and Rural Sciences. The story is timeless. A major event in the history of genetics was the

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Prof. Neil Jones rnj@aber.ac.uk

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  1. Barbara McClintock’s controlling elements: the full story Prof. Neil Jones rnj@aber.ac.uk IBERS - Institute of Biological, Environmental and Rural Sciences

  2. The story is timeless A major event in the history of genetics was the discovery in 1947 that genes could transpose. Published 1951: Cold Spring Harbor Symp Quant Biol 16: 13-47 The story was not believed for 20 years, until restriction enzymes (1970s), made it possible to clone IS elements from bacteria. McClintock was isolated from the Genetics Community for more than 20 years after publishing her work. She was award the Nobel Prize in 1983 1902-1992

  3. Pericarp is maternal tissue Structure of the maize kernel endosperm /aleurone products of fertilisation aleurone layer (3n) - develops pigment endosperm (3n) Embryo 2n pericarp Pigment characters scored without growing seeds

  4. Chromosomes of maize Pachytene stage of meiosis Photo: Bill Sheridan, University of North Dakota

  5. How to make a sticky end without losing anything ? Male meiosis (x=sticky end) A B C C B A A B inverted duplication A x C microspore B C A C B B 1 2 C A 9S A (n) 3 4 B C C x replication / fusion of sticky ends C B A B A C C 1 unbalanced B A B 2 > 90% balanced A 3,4 non-male transmission deficient (due to genetic constitution) Chiasma pachytene metaphase I tetrad

  6. Sticky ends – gametophyte - in plants gametes are produced by mitosis tube nucleus x x x x x x pollen grain mitosis pollen introduced into crosses HOW BEHAVE ?? microspore (n)

  7. Chromatid breakage-fusion-bridge cycle [BFB] Sticky ends from male parent only x x sperm sperm replication and fusion (F) embryo sac x Endosperm- BFB x x EMBRYO Endosperm - BFB HEALING of broken end almost immediately after fertilisation breakage (B) bridge (B) ..based on cytological observations and on the use of markers

  8. Endosperm variegation due to the chromatid BFB c wx (colourless) (amylopectin, red) ♀♀ 9S triploid endosperm X ♂ (purple kernels) C Wx (amylose, blue) Wx C C Wx C C C C Wx x x Wx Wx Wx Replication followed by B-F-B and off-centre breaks c wx c Wx C Wx Reading phenotypes: size of spots = stage of loss “spots within spots” (wx / Wx by iodine stain)

  9. Chromosome breakage-fusion-bridge cycle [BFB] both parents generating sticky ends x x 1 5 fusion at telophase – before replication (prevents chromatid BFB) 2 Zygote no healing 6 prophase 3 anaphase 7 x x either or 4 x x Chromosome BFB cycle continues during early development. Healing >> later in development. Tried use chromosome BFB to induce small internal deficiencies in 9S to study mutations. Endosperm 3 chrs >>independent chromatid BFB cycles WHAT DOES THIS HAVE TO DO WITH TRANSPOSITION?

  10. ‘Earthquake ear’ of 1944 CI =colour inhibitor wd CI Wx x ♂ This cross was made to induce internal deficiencies in 9S. The chromosome BFB was taking place in this F1 early in development, and this triggered a ‘genetic earthquake’. 9S x ♀ wx Wd C 670 KERNELS 590 germinated; 134 died as seedlings; 456 transferred to the field; 73 died; 383 PLANTS - SELFED OR FIXED IN 1945 earthquake ear of 1944 A new type of aleurone variegation appeared. It had a uniform pattern of coloured spots (C) of similar size. It seemed that CIwas being eliminated in some cells at a particular rate, and at a particular stage in development. ONE of the selfed plants, which must have been heterozygous CI//C, gave a few variegated kernels, UNEXPECTEDNO STICKY ENDS INTRODUCED CONTROLLED BREAKAGE she sensed that these were something special – BREAKAGE WAS TAKING PLACE Seeds grown and studied - additional markers (Pale yellow = colourless)

  11. Cytological disturbances 150 plants grown from earthquake ear - fixed for pachytene analysis: • Deficiencies in chromosome 9 • Duplications of 9S • Telocentrics • Isochromosomes • Breaks in chromosomes other than 9 • Inversions • Knob fusions • Plus: • 32 newly arising stable mutants, due to small deficiencies, • and several unstable mutants affecting sectors of the • plant phenotype – controlled, and taking place at different • Times in development Genetic ‘earthquake’

  12. ‘Earthquake ear’ of 1944 CI =colour inhibitor wd CI Wx x ♂ This cross was made to induce internal deficiencies in 9S. The chromosome BFB was taking place in this F1 early in development, and this triggered a ‘genetic earthquake’. 9S x ♀ wx Wd C 670 KERNELS 590 germinated; 134 died as seedlings; 456 transferred to the field; 73 died; 383 PLANTS - SELFED OR FIXED IN 1945 earthquake ear of 1944 New type of aleurone variegation appeared. With a uniform pattern of coloured spots (C) of similar size. It seemed that CIwas being eliminated in some cells at a particular rate, and at a particular stage in development. ONE of the selfed plants, which must have been heterozygous CI//C, gave a few variegated kernels, - UNEXPECTEDNO STICKY ENDS INTRODUCED CONTROLLED BREAKAGE She sensed that these were something special BREAKAGE WAS TAKING PLACE Grown and studied – using additional markers (Pale yellow = colourless)

  13. Discovery of Ds – dissociation locus Breakage without sticky ends – but where was it taking place ? no markers, no BFB Ds Markers: CI colour inhibitor C coloured aleurone c colourless aleurone CI > C > c (dominance) Sh normal endosperm sh shrunken endosperm Bz purple aleurone bz bronze aleurone Wx amylose, blue starch wx amylopectin, red starch Bz CI Sh Wx x ♂ x ♀♀ sh bz wx C CI Bz (Sh Wx) C bz (sh wx) Sectoring not uniform: controlled breakage and loss of all four dominant markers at the same time …. also plant markers .. Pachytene breaks in 9S in some plants, in one of the homologues and acentric fragments seen. Break always at the same site - junction of the euchromatin and heterochromatin – Ds locus. What was Ds?How were breaks controlled in relation to development – some breaks late in development.

  14. Discovery of Ac C ds CI Ds The first clue about control of DS Kernels found without variegation in plants expected with Ds breaks. All progeny were expected to be heterozygous with variegated kernels due to loss of the CI allele: only half variegated – a 1:1 ratio. One of the parents must have been heterozygous for another factor. She called it Activator or Ac. ♀ x ♂ C ds CI Ds C ds Ac CI Ds ac Breeding tests, using appropriate Ds stocks, confirmed that Ac was inherited independently of Ds and acted as a dominant allele in crosses.

  15. Inheritance of Ac C ds ac CI Ds Ac x ac ac C ds CI Ds C ds Ac 1/2 Ds ac CI C ds ac 1/2 Ds CI ac Breeding tests, using appropriate Ds stocks, confirmed that Ac was inherited independently of Ds and acted as a dominant allele in crosses: Ac//ac x Ac//ac 1Ac Ac:2Ac ac:1ac ac Ac//ac x ac//ac 1Ac ac:1ac ac Ac//Ac x ac//ac all Ac ac

  16. Dosage effect of Ac CI Sh Bz Wx Ds Evidence for the controlling effect of Ac came from varying the dosage of Ac in the triploid endosperm: 0, 1, 2, or 3 doses. The dosage of Ac controlled when Ds breakage would take place, but how did frequency of breaks alter in development. ♂ ♀ ♀ C sh bz wx ds + ac ac ac 0 + Ac ac ac 1 + Ac Ac ac 2 + Ac Ac Ac 3

  17. Change of state of Ac Ac CI Sh Bz Wx Ds C sh bz wx several plants x Ac CI Sh Bz Wx Ds C sh bz wx The effect of Ac varied in different plants, different ears of one plant, and different parts of a single kernel. The formation of sectorial kernels, due altered times of breakage, indicated changed forms of Ac – mimicked the Ac dosage effect. Further breeding tests showed that the altered kernels were due to change in the state of Ac, and also a change in the number of Ac elements. Ac controlled the time of breakage of Ds and Ac could change its state Ac CI Sh Bz Wx Ds IdenticalAc alleles from this cross C sh bz wx Sectorial kernel

  18. Transposition of Ds in 1947 Ac C Sh wx Ds ½ ♂ F1 ♀♀ (12) ½ c sh wx ds ac While trying to map Ds in its standard location an unexpected event took place at the C locus. It changed to a new mutable form cm-1. Male parent was Ac/ac Female parent had no Ac or Ds elements. Half kernels expected purple – no Ac Other half variegated with colourless - with Ac. Found in all 12 EARS, but 1/4000 was different. Colour pattern was reversed. Tests indicated: ● cm-1 had reverted to C in this kernel. ●Reversion in chromosome with Ac in male parent in F1. ● Ds breaks present in chromosome with new cm-1locus ● Location of Ds had also moved – inseparable from cm-1 - Ac + Ac

  19. Mutation of the C locus – the interpretation (intellectual leap) The position of Ds had also moved and was inseparable from the new cm-1 locus. The site of chromosome breaks had moved (transposed). Purple spots would only appear if Ac was also present … and …. dosage effect of Ac Evidence consistent with cm-1 arising from transposition of Ds and its insertion into C – moving out from cell clones . Not explained by Ds breakage Ds now had new function > mutation. Other mutable loci later .. Transposition was discovered! (a) Ds C locus purple Ds (b) “footprint” mutant cm-1 locus colourless Ac Ds (c) Ac C

  20. Ds Transposition CI Ds Bz Sh Wx Ac ♂ ♀♀ Ac C sh bz wx C Sh Bz Wx Wx Bz Sub-sectors: C sh Bz Wx C sh bz Wx C sh bz wx Exceptions: Sh Wx Sh Bz Bz CI CI Sh Bz Wx x Sh Sh C sh bz Wx C sh bz wx CI Bz Sub-sectors: C sh bz wx Reveal new position of Ds Wx Wx x twin sectors

  21. Transposition of Ds Ds C Sh Bz Wx With an adequate means of detection it is possible to show that Ds can transpose to numerous other sites within the chromosome complement • (i) insertion into new loci (new mutations) • (ii) kernels with new patterns of variegation • in the absence of Ac the Ds locus is stable, • and can be mapped by recombination analysis Ds could also change its state – NOT SHOWN

  22. Transposition of Ac in early studies Ac was not linked with 9S markers: C sh bz wx CI Sh Bz Wx Ds Ac ♂ x ♀ C Sh ac wx Ds ac C sh bz wx in later crosses linkage was sometimes found: C sh bz wx CI Sh Bz Wx Ds Ac ♂ x ♀ C sh bz wx C Sh Bz wx Ds ac 20% Position varied in different crosses (ears): CI Sh Bz Wx Ds Ac Ac Ac

  23. Transposition of Ac explains some unexpected events Ac//Ac x ac//ac Expected: Ac//ac (usually found) Unexpected: Ac ac ac ac//ac (loss) Ac Ac Ac Ac Ac//Ac (unlinked) Ac Ac Ac//Ac (linked) Ac Ac Ac

  24. The Ac – Ds family of ‘controlling elements’ Ac activates breakage at Ds. Loci may be on different chromosomes. Where did they come from? They were present all the time. The genome shock in the ‘earthquake’ ear activated them from being buried in heterochromatin somewhere in the genome. Ac Ds Ac Ds Ac can promote its own transposition, or that of Ds, to another site either on the same chromosome or on a different one. Ds cannot move unless Ac is present in the same cell. Ac is AUTONOMOUS Ds is NON-AUTONOMOUS Ac Ac Ac Ds Cohesive ends Ac Ds

  25. Cloning McClintock’s elements CAGGGATGAAA Ac - 4563 bp TTTCATCCCTA Exon 1 2 3 4 5 transcription of transposase gene Ds Ds2d1 Ds2d2 Ds6 Changes of state: – insertion into another gene, change of methylation at target site, transposase doubles up as repressor of transposition. Not thought to have role in development. DNA transposons make the genome dynamic: - increase in number if transpose before replication. Transposon promoter may insert next to gene and change its pattern of expression, causing alternative splicing.

  26. Lectures in St. Petersburg • 1995 Advances in B chromosome research • 2000 Physical mapping of plant chromosomes • 2001 Genetically modified crops • Challenging genome integrity • Chromosomes without genes • What is a centromere? • 2006 Order and chaos in the plant nucleus. • 2006 What is a telomere? • 2007 Epigenetics • What is a gene? • 2009 Epigenesis to Epigenetics • Chromosomes without genes revisited. • 2012 McClintock’s controlling elements: the full story Acknowledgements Dynasty Foundation for financial support

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